The structure of dihydrofolate reductase. Identification of methionine residues carboxymethylated by iodoacetate with loss of catalytic activity.

Dihydrofolate reductase from the amethopterin-resistant mutant (strain A) of Streptococcus faecium var. Durans was reacted with iodo[14C]acetate according to three procedures; (a) in the absence of an inhibitor, (b) in the presence of aminopterin, and (c) in absence of inhibitor, but after treatment with unlabeled iodoacetate in presence of aminopterin. The first and last procedures resulted in the loss of approximately 90% of the catalytic activity, whereas in the presence of aminopterin essentially no activity was lost. Peptides were produced from all three labeled proteins by tryptic digestion after citraconylation of the lysine residues. From the amino acid compositions and partial amino acid sequences of these peptides the position of all modified methionines in the sequence was determined. The extent of labeling at each methionine, in enzyme labeled in the different procedures, indicated that methionines 28 and 50 may be at the binding site for inhibitors and that residue 50 is less accessible to iodoacetate than is residue 28. It is likely that carboxymethylation of residue 28 is responsible for the loss of enzyme activity.